(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) World Intellectual PropertyOrganization

International Bureau(10) International Publication Number

(43) International Publication Date1 May 2014 (01.05.2014)

WO 2014/066830 AlP O PCT

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for everyA61K 39/395 (2006.01) A61P 3/04 (2006.01) kind of national protection available): AE, AG, AL, AM,A61K 48/00 (2006.01) A61P 3/00 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,

BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,(21) International Application Number: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,

PCT/US20 13/066932 HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,

(22) International Filing Date: KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,

25 October 2013 (25.10.201 3) MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,

(25) Filing Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,

(26) Publication Language: English TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,ZW.

(30) Priority Data:61/795,815 25 October 2012 (25. 10.2012) U S (84) Designated States (unless otherwise indicated, for every

kind of regional protection available): ARIPO (BW, GH,(71) Applicant: DEPARTMENT OF VETERANS AFFAIRS GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,

[US/US]; 810 Vermont Avenue, NW, Washington, District UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,of Columbia 20420 (US). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,

EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ , LT, LU, LV,(72) Inventors: KISHORE, Bellamkonda K.; 810 VermontMC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,

Avenue, NW, Washington, District of Columbia 20420TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,

(US). ZHANG, Yue; 810 Vermont Avenue, NW, WashKM, ML, MR, NE, SN, TD, TG).

ington, District of Columbia 20420 (US). ECELBAR-GER, Carolyn M.; 810 Vermont Avenue, NW, W ashing Published:ton, District of Columbia 20420 (US).

— with international search report (Art. 21(3))(74) Agents: HADDACH, Aubrey, A. et al; Procopio Cory

Hargreaves & Savitch LLP, 525 B Street, Suite 2200, SanDiego, California 92101 (US).

(54) Title: COMPOSITION AND METHODS FOR THE PREVENTION AND TREATMENT OF DIET-INDUCED OBESITY

Fig, 1A: Change In Body Weight of Wild Type (WT) and P2Y : knockout jKO) Mice fedRegular Diet (CNT) or High-Fat Diet HFD) for 1 Weeks with ad libitum Access to Food

and Drinking Water

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o(57) Abstract: Blunting the activity of the P2Y2 receptor results in a resistance to diet-induced obesity, an increased metabolic rate,and a better glucose tolerance. Compounds that inhibit the puringeric P2Y2 receptor are useful for treating disorders associated withdiabetes, treating obesity, and increasing metabolism (e.g., fatty acid metabolism).

COMPOSITION AND METHODS FOR THE PREVENTION AND TREATMENT OFDIET-INDUCED OBESITY

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No.

61/795,815, filed October 25, 2012, which is incorporated by reference in its entirety.

GOVERNMENT RIGHTS

[0002] The present invention was made with government support under Department of

Veterans Affairs Merit Review Program, Grant No. BLRD 000591, and the facilities and

resources at the VA Salt Lake City Health Care System. The Government has certain rights

to this invention.

FIELD OF THE INVENTION

[0003] Blunting the activity of the P2Y2 receptor results in a resistance to diet-induced

obesity, an increased metabolic activity, and a better glucose tolerance. Therefore,

compounds that inhibit the puringeric P2Y2 receptor are useful for treating diet-induced

obesity and metabolic disorders associated therewith, such as diabetes mellitus.

BACKGROUND OF THE INVENTION

[0004] Obesity is one of the major public health problems of the 21st century with

significant consequences on health of the individuals and impact on the national economies.

The most common cause of obesity is diet-induced, i.e., consumption of more calories than

one can metabolize. In view of the rapidly growing number of obese people throughout the

world, the global market for anti-obesity drugs is estimated to reach $3.4 billion by 2016.

However, currently available drugs for the treatment of obesity have significant limitations in

terms of safety, efficacy or both. Thus, there is a high unmet need for safe and efficacious

medications for the prevention or treatment of obesity. This invention discloses a potential

drug target for the treatment of diet-induced overweight or obesity. This approach involves

increasing the metabolism of consumed excess calories, which may prove to be safer or

tolerable than the other two approaches, namely suppression of the appetite or interfering

with the absorption of fat in the intestines. It is possible to develop orally effective chemical

compounds that selectively act at the disclosed target as potential anti-obesity drugs.

[0005] According to the World Health Organization, a Body Mass Index (BMI) > 25 is

overweight, and a BMI > 30 is obesity. Overweight and obesity are no more the problems of

high-income countries of the world. They are rapidly rising in low- and middle-income

countries.

[0006] In 2008 the World Health Organization estimated that globally more than 1.4

billion adults were overweight; and the 2010 estimates showed that more than 40 million

children under five years are overweight. Overweight and obesity are the fifth leading risk

for global deaths, accounting for 2.8 million deaths each year. In fact, overweight and

obesity are linked to more deaths worldwide than underweight.

[0007] Raised BMI is a major risk factor for cardiovascular and musculoskeletal

disorders, and certain types of cancers, such as breast and colon cancers. In fact, 44% of the

diabetic burden, 23% of the ischemic heart disease burden and between 7% and 14% of

certain cancer burdens are attributable to overweight and obesity.

[0008] The global economic burden of overweight and obesity are not well established.

But the numbers are more accurately available for the United States. Over the past 50 years

the prevalence of obesity in the USA has almost tripled from 13% to 34%, with the

percentage of Americans who are extremely or "morbidly" obese rising from 0.9 to 6. The

estimated annual medical costs due to obesity are about $190 billion. The indirect costs due

to overweight and obesity are also sky rocketing. For example, it is estimated that airlines

incur about $5 billion annually for additional jet fuel needed to fly obese or heavier people as

compared to the fuel needed to fly the same number of people based on the body weights

prevalent in 1960s. Automobiles also consume extra gasoline amounting to about $4 billion

to transport heavier people in the USA. The annual costs due to absenteeism among over

weight and obese people are also very high. In fact, according to a study, the costs of obesity

exceed those of smoking.

[0009] According to the World Health Organization, the fundamental cause of obesity

and overweight is energy imbalance between calories consumed and calories expended. In

recent decades globally there has been an increase in caloric intake and a decrease in the

physical activity, driving the incidence of obesity high. Although supportive environments

and communities resulting in consumption of healthy foods and regular physical activity are

the best to prevent overweight and obesity, these do not happen often, in part because of the

complex nature of modern day living.

[0010] Currently used modalities as well as the pipeline drugs for the prevention and/or

treatment of obesity fall into the following three categories: (1) drugs that suppress appetite

by acting on the brain (e.g., sibutramine (i.e., Reductil® and/or Meridia®); (2) drugs that boost

body's metabolic rate (e.g., cannaboid receptor antagonists and Metformin); and (3) drugs

that interfere with the body's ability to absorb specific nutrients in food, such as fat (e.g.,

Orlistat®, Xenical®, and/or Alii®) .

[0011] Drugs that suppress the appetite by acting on the brain have severe side effects

which are neurological and/or psychological in nature. For example, these drugs carry a risk

of high blood pressure, faster heart rate, palpitation, restlessness, agitation, insomnia etc.

Due to these limitations, drugs that suppress appetite have been withdrawn from the market

by the FDA and approval of newer drugs has been made more stringent. Drugs that increase

the body's metabolic rate have their own limitations, such as lack of consistent effect

resulting in sustained loss of weight. Drugs that block absorption of dietary fat, cause oily

stools (steatorrhea), abdominal pain and flatulence.

[0012] In view of the side effects with these therapies, combination therapies that target

more than one pathway have been developed. However, these also have severe side effects

and limitations, despite their efficacy in short-term treatment. Accordingly, there is a high

unmet need for safe and effective medications for the prevention or treatment of overweight

and obesity, which is expected to drive the anti-obesity market growth in the future. The

invention described herein seeks to meet this need.

SUMMARY OF THE INVENTION

[0013] Provided herein are methods for selectively blocking the activity of the P2Y2

purinergic receptor for the prevention and/or treatment of diet-induced overweight or obesity.

It was discovered that the genetic deletion of P2Y2 receptor results in resistance to the

development of high-fat diet-induced obesity and the associated physical and molecular

alterations in mice. The results described also suggest that the observed resistance of the

P2Y2 receptor in knockout mice was not due to reduced consumption of high-fat diet or lack

of absorption of fat in the intestines, but may be due to the ability of the knockout mice to

metabolize or "burn" the extra calories consumed in the diet more efficiently than the wild

type mice. Accordingly, targeting P2Y2 receptor with specific pharmacological agents or

drugs is a safe approach to prevent or treat diet-induced overweight or obesity.

[0014] In various embodiments, provided herein are methods for treating or preventing

diet-induced obesity in a subject by administering an agent that blunts the expression or

activity of the P2Y2 receptor in the subject.

[0015] In some specific embodiments, the agent is a fusion protein or an agent that

inhibits the P2Y2 receptor. In specific embodiments, the agent is a selective P2Y2 receptor

antagonist, including but not limited to a non-nucleic acid small organic molecule.

[0016] In other embodiments, the agent is an oligonucleotide. In specific embodiments,

the oligonucleotide is an antisense oligonucleotide, a ribozyme or an inhibitory RNA,

including but not limited to a small inhibitory RNA (siRNA).

[0017] In other embodiments, provided herein are methods for increasing energy

metabolism in a cell by contacting the cell with an agent that blunts the expression or activity

of a P2Y2 receptor, thereby increasing the metabolism in the cell. In specific embodiments,

the cell is an adipocyte cell.

[0018] In specific embodiments, the agent is a fusion protein, polypeptide, a small non-

nucleic acid organic molecule, a small inorganic molecule or an oligonucleotide, including

but not limited to an antibody, an antisense oligonucleotide, a ribozyme, or an inhibitory

RNA, including but not limited to a small inhibitory RNA (siRNA).

[0019] Also provided herein are methods for increasing energy metabolism in an

adipocyte cell in a subject that is at risk for or suffering from a disorder related to glucose

metabolism, the method comprising administering to the subject an agent that inhibits

expression or activity of a P2Y2 receptor, thereby increasing the metabolism in the cell.

[0020] In various specific embodiments, the agent is a polypeptide, a small non-nucleic

acid organic molecule, a small inorganic molecule, an antibody, an antisense oligonucleotide,

a ribozyme or an inhibitory RNA, including but not limited to a small inhibitory RNA

(siRNA).

[0021] Provided herein is also a method for identifying a candidate agent that modulates

expression or activity of the P2Y2 receptor, comprising: (a) providing a sample comprising

the P2Y2 polypeptide or a nucleic acid encoding the polypeptide; (b) contacting the sample

with a test compound under conditions in which the polypeptide is active, the nucleic acid is

expressed, or both; (c) evaluating expression or activity of the P2Y2 polypeptide in the

sample; and (d) comparing the expression or activity of the P2Y2 polypeptide of (c) to the

expression or activity of the P2Y2 polypeptide in a control sample lacking the test compound,

wherein a change in the P2Y2 polypeptide expression or activity indicates that the test

compound is a candidate agent that can modulate the expression or activity of the P2Y2

polypeptide.

[0022] In various specific embodiments, the candidate agent selectively inhibits the P2Y2

receptor or is a fusion protein

[0023] Other features and advantages of the present invention will become more

readily apparent to those of ordinary skill in the art after reviewing the following detailed

description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The details of the present invention may be gleaned in part by study of the

accompanying drawings, in which:

[0025] FIGURE 1 illustrates the changes in the body weights and P2Y2 knockout and

wild-type mice fed regular diet or high-fat diet for 16 weeks.

[0026] FIGURE 2 shows the size of one of the highest weighing wild-type mouse with

an average weighing P2Y2 knockout mouse from the high-fat diet groups.

[0027] FIGURE 3A illustrates the food intake (g/day or g/g body weight) of the mice

after 4 weeks of the Experimental Period.

[0028] FIGURE 3B illustrates the food intake (g/day or g/g body weight) of the mice

after 12 weeks of the Experimental Period.

[0029] FIGURE 4A illustrates the water intake (ml/day or ml/g body weight) of the mice

after 4 weeks of the Experimental Period.

[0030] FIGURE 4B illustrates the water intake (ml/day or ml/g body weight) of the

mice after 12 weeks of the Experimental Period.

[0031] FIGURE 5A illustrates the urine output (ml/g body weight) and urine osmolality

(mOsm/Kg) after 4 weeks of the Experimental Period.

[0032] FIGURE 5B illustrates the urine output (ml/g body weight) and urine osmolality

(mOsm/Kg) after 12 weeks of the Experimental Period.

[0033] FIGURE 6A illustrates the appearance of abdominal fat in a (a) wild type mouse

on a regular diet, (b) wild type mouse on a high-fat diet, and (c) P2Y2 knockout mouse on a

high-fat diet, at the time of euthanasia.

[0034] FIGURE 6B is a representative picture of white fat from a high-fat diet wild type

mouse (left) and knockout mouse (right).

[0035] FIGURE 6C is a representative picture of brown fat from a high-fat diet wild

type mouse (left) and knockout mouse (right).

[0036] FIGURE 7A illustrates the terminal weight of white adipose tissue (mg and

mg/20g body weight).

[0037] FIGURE 7B illustrates the terminal weight of brown adipose tissue (mg and

mg/20g body weight).

[0038] FIGURE 8 illustrates the terminal weight of the liver and kidney (mg/20g body

weight) in the mice after 16 weeks of the Experimental Period.

[0039] FIGURE 9 provides illustrative samples of the fecal matter in high-fat diet fed

wild type mice (left) and knockout mice (right) during the 16 week Experimental Period.

[0040] FIGURE 10 provides the results of a Glucose Tolerance Test during the 14th

week of the Experimental Period.

[0041] FIGURE 11 provides the results of a Serum Insulin assay (left) and a Serum

Leptin assay (right) after 16 weeks of the Experimental Period.

[0042] FIGURE 12 provides the results of a Serum Adiponectin assay (left) and a Serum

Free Fatty Acid assay (right) after 16 weeks of the Experimental Period.

[0043] FIGURE 13 provides the results of a Serum Triglycerides assay (left) and a

Serum Glycerol assay (right) after 16 weeks of the Experimental Period.

[0044] FIGURE 14 provides Messenger RNA Expression level of PPAR in the Brown

Fat (left) and the Messenger RNA Expression level of UCP in the Brown Fat (right).

[0045] FIGURE 15 provides the Messenger RNA Expression level of Inflammatory

Cytokines and Cell Surface Molecules (TNF-a, IL-6, MCP-1, CCR2, CD68, F4/80) in the

White Fat (left) and the Messenger RNA Expression level of Insulin Receptor Substrate

Isoforms (IRS 1, IRS 2) the White Fat (right).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0046] After reading this description it will become apparent to one skilled in the art how

to implement the invention in various alternative embodiments and alternative applications.

However, although various embodiments of the present invention will be described herein, it

is understood that these embodiments are presented by way of example only, and not

limitation. As such, this detailed description of various alternative embodiments should not

be construed to limit the scope or breadth of the present invention as set forth in the appended

claims.

[0047] As shown herein, it was discovered that mice with genetic deletion of P2Y2

receptor consume the same amount of high-fat diet as their syngeneic wild type mice do, and

do not excrete fatty stools (steatorrhea), yet do not become obese. This suggests that P2Y2

receptor is a potential target for the development of efficacious and safer anti-obesity drugs.

One possible reason for this phenomenon is that purinergic P2Y2 receptor is involved in

energy metabolism and so its genetic deletion significantly protects against high-fat diet-

induced obesity. Accordingly, purinergic antagonism may prove to be a very advantageous

invention over the current state-of-the-art.

[0048] This is further supported by the fact that one promising line of future anti-obesity

drugs is based on the development of ribonucleic acid interference (RNAi) to silence RIP 140

gene, a nuclear co-repressor that regulates fat accumulation. This line of drug development

was prompted by the observation that mice with genetic deletion of RIP 140 exhibit a lean

profile throughout their life, and are resistant to diet-induced obesity, and show an enhanced

metabolic rate. CytRx Corporation is currently developing RNAi therapeutics against this

drug target (RIP 140) for the treatment of obesity and type 2 diabetes. Conversely, disruption

of the molecular interaction between SMRT, a nuclear hormone receptor co-repressor and its

receptor partner leads to increased adiposity and decreased metabolic rate.

[0049] The novel findings described herein open the possibility to develop an entirely

new line of drugs that target the molecular interactions between G protein-coupled receptors

and their downstream regulatory effectors to prevent or treat diet-induced obesity. Thanks to

the availability of gene knockout mice that carried these molecular disruptions in vivo

throughout their lifespan, researchers are able to assess the efficacy and safety of this

promising approach in live mice.

[0050] As discussed in detail herein, it was discovered that genetic deletion of P2Y2

receptor causes resistance to diet-induced obesity. The data indicates that this effect is likely

related to increased metabolic rate and the effect is associated with better glucose tolerance as

well as a sign of better balance between energy intake and expenditure.

[0051] The invention described herein has potential practical and commercial

applications to develop a new line of anti-obesity drugs that are safe and efficacious. This

can be achieved by either designing and developing P2Y2 receptor selective antagonists

(chemical molecules) or RNAi to silence the P2Y2 receptor gene.

EXAMPLES

Example 1; Mice That Are Genetically Deficient In P2Y2 Receptor

[0052] A bench prototype was generated in mice that are genetically deficient in P2Y2

receptor. These mutant mice apparently do not suffer from diseases, have no pathological

lesions and live healthy. Although one study showed that these mice develop salt-resistant

hypertension, the rise in blood pressure was modest, and these mice do not develop

complications of hypertension, such as kidney disease. See., Rieg et ah, 2007, FASEB J

21:3717-3726.

[0053] Hence, targeting purinergic signaling for the treatment of obesity is relatively safe

as compared to the current state- of-the-art. Any potential increase in blood pressure can be

effectively controlled with combination therapy.

Example 2; Wild Type and P2Y2 Receptor Knockout Study

[0054] A study was performed on wild type and P2Y2 receptor knockout mice (both in

B6D2 genetic background). Breeders of these mice were originally obtained from Dr.

Beverly Koller of the University of North Carolina at Chapel Hill, Chapel Hill, NC.

[0055] Obesity is associated with deranged sodium homeostasis leading to hypertension

among other pathologies. Hence, this high-fat diet study was conceived to examine the role

of P2Y2 receptor in obesity-induced alterations in water and sodium handling by the kidney,

leading to hypertension. Since kidney is one of the vital organs affected by obesity, we

posited that any new information obtained may help us to address more complex questions,

such as the course of lithium-induced nephrogenic diabetes insipidus in obese patients and

the effect of lack of purinergic signaling. However, during the study we observed that P2Y2

receptor knockout mice were resistant to the diet-induced obesity, which forms the basis for

this invention.

Experimental Study Parameters

[0056] Breeding colonies were established in the Veterinary Medical Unit (VMU) of the

VA Salt Lake City Health Care System. Mice bred were identified by Polymerase Chain

Reaction ("PCR") on DNA extracted from the tail clippings, and all mice were tracked by

implanting microchip transponders under the skin. Age matched adult wild type and P2Y2

receptor knockout (KO) mice were randomly divided into two sub-groups for each genotype

and were fed regular rodent chow (10% of total energy as fat) or high-fat diet (60% or total

energy as fat) for 16 weeks as shown below. The high-fat diet was purchased from the

Research Diets, Inc. (New Brunswick, NJ).

[0057] All mice had free access to drinking water throughout the experimental period.

Food intake and body weights were monitored on regular basis. Twenty-four hour urine

samples were collected periodically by placing the mice in metabolic cages with free access

to food and drinking water. Feces samples of all groups of mice were collected periodically.

During the 14 th week tolerance of the mice to an acute load of glucose was assessed by

carrying out glucose tolerance test ("GTT"). All mice were euthanized at the end of the

experimental period and samples of blood, liver, kidney, pancreas, spleen and whole white

and brown adipose tissues were collected for analysis in the laboratory.

[0058] The GTT tests were performed as described by Takahashi et al (2011). Briefly,

after a fasting period of about 12 hours, the mice received an intraperitoneal injection of

sterile glucose solution (1.5 g/kg body w ). Tiny droplets of blood (~ 5 µΐ) were obtained in

conscious mice by gently pricking the dorsal pedal vein with a fine needle. Using these

droplets, blood glucose levels were determined with a clinical glucometer. Blood samples

were collected prior to (0 min), and 30, 60, 90 and 120 min after intraperitoneal injection of

glucose solution.

[0059] Twenty-four hour urine output was recorded, and the osmolalities of clear urine

samples were determined by vapor pressure method on an osmometer (Wescor, Logan, UT).

Insulin, leptin, adiponectin, triglycerides and glycerol levels in the serum samples were

determined by using commercial ELISA or EIA kits (Crystal Chem, Inc., Downers Grove, IL

or Cayman Chemical Co., Ann Arbor, MI). Weights of total brown or white adipose tissues

were determined for each mouse. Total liver and kidney weights were also determined.

[0060] Liver tissue samples were analyzed for metabolic changes in the University of

Utah Metabolomic Core Facility using gas chromatography-mass spectrometry (GC-MS).

For this analysis, about 20 mg of liver tissue from each mouse (N = 6 mice/group) was used.

The liver tissue was flash frozen in liquid nitrogen at the time of euthanasia and then stored at

-80°C. In the core facility, the tissue was mechanically disrupted using a bead mill, and

metabolites were extracted by a solvent. After evaporating the solvent, the dried samples

were suspended in O-methoxylamine hydrochloride (MOX) and further processed for GC-

MS. GC-MS data was collected using MassLynx 4.1 software (Waters).

[0061] A two-step process was employed for data analysis, a targeted followed by non-

targeted analysis. For the targeted approach known metabolites were identified and their

peak areas were recorded using QuanLynx. For the non-targeted approach peak picking and

analysis was performed using MarkerLynx. Principle components analysis (PCA) and partial

least squares-discriminate analysis (PLS-DA) was performed using SIMCA-P 12.0

(Umetrics, Kinnelon, NJ). Metabolite identity was established using a combination of an in-

house metabolite library developed using pure standards and the commercially available

NIST library. Not all metabolites were observed using GC-MS.

[0062] Quantitative data are presented as mean ± sem. Comparisons among the means of

multiple groups were made by one-way analysis of variance (ANOVA), followed by the

assessment of statistical significance by Tukey-Kramer Multiple Comparison test or

Boneferroni test for selected pairs. Differences between the means of two groups were

determined by unpaired t-test or Mann-Whitney nonparametric method. P values less than

0.05 were considered significant. GraphPad Instat® software (GraphPad Software, Inc., La

Jolla, CA) was used for statistical analysis.

Study Results

[0063] Figure 1 illustrates the changes in the body weights in (WT) and P2Y2 knockout

(KO) mice fed regular diet or high-fat diet for 16 weeks. As shown in Figure 1A, the WT

mice had modestly, but significantly higher body weights even on day 0 as compared to the

age-matched KO mice. Following high-fat diet feeding, the body weight or WT mice

steadily increased starting at 2 weeks. In contrast, body weights of high-fat diet fed KO mice

essentially remained similar to the regular diet-fed KO mice throughout the study period.

Figure IB and Figure 1C show the terminal (16 weeks) body weights of the high-fat diet fed

WT vs. high-fat diet fed KO mice. The differences between these two groups are striking

when analyzed as weights of individual mice using scattergraph (Figure IB) or as box plots

(Figure 1C).

[0064] Figure 2 shows a picture of mice to compare the size of one of the highest

weighing WT mice from the high-fat diet group (WT-HFD) with an average weighing KO

mouse from the high-fat diet group (KO-HFD). In order to exclude the possibility that the

differences observed in body weights of the WT and KO mice was due to differences in their

food and/or water intake, the food and water intake of the mice at two different time points

was determined (see below).

[0065] Figure 3A shows food intake as g/day/mouse after 4 weeks. As shown in this

figure, the high-fat diet fed WT mice consumed significantly less amount of food as

compared to the regular diet fed WT mice. However, there was no difference in the food

intake between the WT or KO mice when high-fat diet was fed to them.

[0066] Figure 3B shows food intake in high-fat diet fed WT or KO mice after 12 weeks.

The left panel presents the data as g/day/mouse, whereas the right panel shows the data as g/g

body weight per day. Notably, at this time point the KO mice were consuming significantly

higher amount of food, despite the fact that their body weights were significantly lower as

compared to the body weights of the high-fat diet fed WT mice (see, Figure 1). Thus, the

observed lack of gain in the body weight in high-fat diet fed KO mice was not due to

consumption of lesser amount of food. In fact, food consumption in these mice is relatively

higher when normalized to the body weight.

[0067] Figure 4A shows water intake as ml/day/mouse after 4 weeks. Although there

were no significant differences between the genotypes, high-fat diet significantly reduced

water intake in both genotypes. Figure 4B shows water intake in high-fat diet fed WT and

KO mice after 12 weeks. The left panel presents data as ml/day/mouse, whereas the right

panel shows data as ml/g body weight per day. Similar to the food consumption, the KO

mice were drinking significantly higher amount of water relative to their body weight, despite

the fact that their body weights were significantly low as compared to the body weights of the

high-fat diet fed WT mice (see, Figure 1).

[0068] Figure 5A shows urine output and urine osmolality after 4 weeks in WT and KO

mice fed regular or high-fat diets. As shown in the left panel, high-fat diet caused significant

decrease in urine output in both genotypes. However, high-fat diet did not have any effect on

the urine osmolality in WT or KO mice. Figure 5B shows urine output and urine osmolality

in high-fat diet fed WT and KO mice after 12 weeks. Interestingly at this time point the urine

output when expressed as ml/g body wt/day was modest, but significantly higher in the KO

mice (left panel). However, no differences were noted in urine osmolality between these two

groups after 1 weeks.

[0069] Figure 6A shows representative pictures of the appearance of abdominal fat at the

time of euthanasia. Panel 6Aa shows the appearance of normal content and disposition of

abdominal fat in a WT mouse fed regular diet, which is similar to KO mice fed a regular diet

(not shown here). In contrast, high-fat diet fed WT mice have much higher amounts of

abdominal fat (Figure 6Ab) as compared to the high-fat diet fed KO mice (Figure 6Ac).

[0070] In mammals, fat or adipose tissue exists in two distinctive forms - the white and

brown adipose tissues. White adipose tissue is a storage form of energy and accumulates

excessive calories consumed as fat droplets, usually found around the abdominal wall or the

belly. White adipose tissue burns slowly, and is linked to the risk for diabetes and heart

diseases. Conversely, the brown fat, which is rich in mitochondria and capillaries (hence the

color), burns calories to generate heat. Brown fat is abundant in newborns and hibernating

mammals, whereas the white fat is abundant in adults. Figure 6B and Figure 6C are

representative pictures of white or brown fat obtained from the high-fat diet fed WT or KO

mice. The difference in the masses of these two types of tissues between the genotypes was

quite obvious.

[0071] Figure 7A shows the terminal weights of white adipose tissue in the WT and KO

mice fed regular or high-fat diet. As shown, the WT mice fed high-fat diet had a 2 to 3-fold

higher amount of white fat as compared to the regular diet fed WT mice, depending on

whether the fat content was expressed as mg/mouse or mg/20 g body weight. On the other

hand, the high-fat diet feeding had no significant effect on the white adipose tissue in the KO

mice. Figure 7B shows the terminal weights of brown adipose tissue in WT and KO mice fed

high-fat diet. Similar to the white adipose tissue, high-fat diet feeding had a significant effect

on brown adipose tissue only in the WT mice.

[0072] Since the liver and kidney are two vital organs affected by obesity, their weights

were recorded as a function of body weight at the time of euthanasia. As shown in Figure 8,

in high-fat diet fed WT mice the weights of both liver and the kidney were significantly low

when adjusted for the body weight. In contrast, a high-fat diet did not have any effect on the

weights of the liver and kidney in KO mice.

[0073] If the apparent resistance of the KO mice to high-fat diet induced increase in body

weight and accumulation of body fat is due to defective absorption of fat in the intestines,

then one would expect steatorrhea in these mice. Steatorrhea is a condition where the feces

contain partially digested fat as oil. The feces are not well formed, and have an oily

appearance and foul smell. Hence, the fecal matter in the high-fat diet fed WT and KO mice

was monitored. Figure 9 shows representative profiles of the fecal matter in high-fat diet fed

wild type (WT-HFD) and knockout (KO-HFD) mice collected during the last week of

experimental period (16 weeks). As illustrated in this figure, both WT and KO mice fed

high-fat diet have well-formed, and non-oily fecal matter. These observations rule out the

possibility of lack of absorption of fat in the intestines in KO mice.

[0074] Glucose tolerance or the ability to metabolize a load of glucose is impaired in

obese subjects. And obesity is a known risk factor for the development of type II diabetes,

where the subjects develop resistance to insulin and thus cannot metabolize glucose

efficiently, despite high blood insulin levels. To examine whether the high-fat diet induced

increase in body weight in the wild type mice is associated with glucose intolerance, a

Glucose Tolerance Test (GTT) using a standard protocol was performed.

[0075] Figure 10 shows the results of the GTT in different groups of mice. As shown in

the left panel of Figure 10, at 30 min after an acute load of glucose, the high-fat diet fed WT

mice showed the highest blood level of glucose, about 400 mg/dl. The corresponding blood

glucose level in high-fat diet fed KO mice was significantly low. In fact at all the time points

following an acute glucose load, the high-fat diet fed KO mice had significantly lower blood

glucose levels as compared to the high- fat diet fed WT mice (Figure 10, right panel).

[0076] Notably, the baseline (0 min) blood glucose levels in the WT mice were modest,

but significantly elevated as compared to the KO mice (Figure 10, right panel). Furthermore,

at 30 min time point following an acute load of glucose, the differences among the four

groups were very obvious, with the two WT groups having higher blood glucose levels as

compared to the two KO groups. Similar to the high-fat diet fed WT and KO mice, the

differences in blood glucose levels between the regular diet fed WT and KO mice were also

wide apart at 30 min time point, indicating that KO mice are inherently efficient in

metabolizing a load of glucose faster.

[0077] Insulin, released from the beta-cells of islets of Langerhans in the pancreas in

response to increasing blood glucose levels (e.g., following a meal), is the key hormone in

regulating the blood glucose levels by inducing uptake of glucose by cells and its subsequent

metabolism. In type II diabetes, the cells develop resistance to the action of insulin with the

result the subjects have both high blood glucose and high blood insulin levels. The left panel

of Figure 11 shows terminal serum insulin levels in the blood samples collected at the time of

euthanasia. As illustrated in this figure, following high-fat diet feeding for 16 weeks, the WT

mice had more than 2-fold increase in serum insulin. The insulin levels in the KO mice were

not affected by high-fat diet feeding. These results correlate well with the pattern of GTT

results obtained.

[0078] Leptin is a peptide hormone elaborated mainly by white adipose tissue, although

brown adipose tissue and other organs also produce leptin. The levels of circulating leptin

are proportional to the total amount of the fat in the body. In general, leptin is considered to

play a key role in regulating energy intake and energy expenditure, including appetite and

metabolism. Leptin acts on the hypothalamus and inhibits appetite. Hence, the absence of

leptin leads to uncontrolled food intake resulting in obesity, e.g., ob/ob mutant mice that lack

leptin.

[0079] When these mutant mice are treated with leptin injections, they lose excess body

fat and return to normal body weight. However, despite these properties of leptin, obese

subjects often develop resistance to leptin so their appetite is not reduced by high circulating

leptin levels. This is because of leptin desensitization due to high circulating leptin levels in

obese subjects. In view of this, serum levels of leptin in the mice were determined. As

shown in the right panel of Figure 11, high-fat diet feeding for 16 weeks caused more than a

3-fold increase in the serum leptin levels in WT mice. The increase in serum leptin levels in

high-fat diet fed KO mice was modest.

[0080] Adiponectin is a protein hormone produced exclusively from adipose tissue (and

also from placenta during pregnancy). Adiponectin modulates a number of metabolic

functions, such as glucose metabolism and fatty acid catabolism. Adiponectin also plays

roles in the suppression of metabolic derangements, such as those occurring due to type II

diabetes mellitus, obesity, atherosclerosis, as well as others. In general, adiponectin is

considered as an independent risk factor for metabolic syndrome. Similar to leptin, the

weight reducing effects of adiponectin are mediated via brain. In view of this, the serum

adiponectin levels in the blood samples collected was determined at the time of euthanasia,

i.e., after 16 weeks of feeding the respective diets.

[0081] As shown in Figure 12 left panel, the serum adiponectin levels are modestly, but

significantly elevated in high-fat diet fed WT mice, but not in KO mice. The increase in

serum adiponectin in the WT mice may be due to higher fat content in the body, and also in

response to the increase in the body weight. However, similar to higher serum leptin levels in

these mice, the higher adiponectin levels did not counter the high-fat diet-induced increase in

body weight in WT mice. Since adiponectin is involved in fatty acid catabolism, the serum

free fatty acids (FFA; Figure 12, right panel), serum triglycerides (TG; Figure 13, left panel)

and serum glycerol (Figure 13, right panel) were determined. As shown, the serum FFA

levels were not significantly different among the four groups of mice, although high-fat diet

had a tendency for higher levels of serum FFA in both genotypes. Interestingly, serum TG

levels were significantly higher in KO mice as compared to the WT mice, irrespective of the

dietary regimen, and dietary regimen itself did not have any effect in either genotype. In

contrast, a high-fat diet triggered significantly higher (about 2-fold) serum glycerol levels in

both genotypes.

[0082] Further insights into the metabolic profiles of the WT and KO mice and the effect

of high-fat diet feeding, were obtained by performing a metabolomics analysis of the liver

tissue. Table 1 below provides the data obtained for 23 carbohydrate metabolites, 20 protein

metabolites, 22 lipid metabolites and 5 nucleic acid metabolites.

Table 1: Metabolomic Analysis of Liver Tissue by GC-MSGC-MS data presented here are in arbitrary units and expressed as mean ± semMetabolites shown in orange background with an asterisk on the right end of the

line haveinteresting or significant trends with respect to genotype or dietary regimen or

both.

WT-CNT WT-HFD KO-CNT KO-HFDCarbohydrate Metabolites

Lactic Acid 687 ± 89 556 ± 59 559 ± 59 585 ± 64Pyruvic Acid 159 ± 34 129 ± 9 110 ± 9 118 ± 6

Glycerol 1.26 ± 1.00 0.69 ± 0.06 3.82 ± 0.24 2.85 ± 0.78 *

Glyceric Acid 558 ± 44 763 ± 79 475 ± 32 941 ± 49 *

Citric Acid 35.3 ± 3.1 34.2 ± 4.4 34.4 ± 3.8 37.0 ± 5.9

Aconitic Acid 7.08 ± 1.38 3.08 ± 0.74 3.12 ± 0.21 2.85 ± 0.43

Isocitric Acid 6.03 ± 1.25 9.65 ± 3.94 5.06 ± 0.21 5.58 ± 0.67

2-Ketoglutaric Acid 1.99 ± 0.57 1.97 ± 0.31 1.08 ± 0.37 1.61 ± 0.24

Succinic Acid 2463 ± 291 2657 ± 293 2402 ± 332 3130 ± 340

Fumaric Acid 355 ± 14 327 ± 45 279 ± 50 330 ± 36

Malic Acid 709 ± 49 753 ± 110 558 ± 73 819 ± 100

2-Hydroxy glutarate 4.06 ± 0.34 4.29 ± 0.48 1.72 ± 0.44 3.09 ± 0.39

2-Aminoadipic Acid 5.70 ± 1.02 9.11 ± 1.54 7.84 ± 1.85 14.72 ± 4.25 *

22570 ±Glucose 21885 ± 853 1575 20706 ± 587 20302 ± 253

Fructose 466 ± 62 536 ± 60 442 ± 47 535 ± 450.287 ± 0.395 ± 0.222 ± 0.321 ±

Ribose 0.019 0.079 0.01 1 0.094

Galactose or isomer 2605 ± 283 1942 ± 112 1994 ± 166 1571 ± 49

Glucose- 1-Phosphate 204 ± 22 194 ± 18 338 ± 27 214 ± 25

Glucose-6-Phosphate 1009 ± 106 1025 ± 161 1922 ± 142 1285 ± 149

Sorbitol 19.9 ± 3.6 12.5 ± 1.1 22.2 ± 4.0 6.5 ± 1.0

Galacitol 289 ± 55 337 ± 15 245 ± 40 348 ± 52

Disaccharide 5 126 ± 1454 5581 ± 743 3866 ± 859 1474 ±314

Xylulose-5-monophosphate 24.3 ± 1.9 20.1 ± 3.5 22.5 ± 1.5 23.2 ± 1.6

WT-CNT WT-HFD KO-CNT KO-HFD

Protein Metabolites

Lysine 40.3 ± 16.3 41.2 ± 13.2 54.9 ± 31.6 50.0 ± 8.6

Valine 417 ± 6 1 422 ± 4 1 412 ± 92 414 ± 28

Leucine 1798 ± 301 1749 ± 154 1659 ± 401 1761 ± 134

Isoleucine 1047 ± 152 1049 ± 92 1002 ± 247 997 ± 123

Threonine 166 ± 29 167 ± 1 1 141 ± 25 211 ± 14

Glycine 582 ± 60 217 ± 33 486 ± 74 171 ± 18

Serine 5 1 ± 9 34 ± 4 39 ± 5 36 ± 5

Alanine 1037 ± 94 826 ± 128 989 ± 179 1230 ± 100

Glutamic Acid 104 ± 18 123 ± 12 92 ± 14 187 ± 32

Glutamine 2891 ± 208 2470 ± 318 1835 ± 258 2783 ± 86

Proline 683 ± 116 776 ± 104 728 ± 174 1835 ± 261

Aspartic Acid 24.7 ± 3.3 29.0 ± 3.6 25.0 ± 3.7 36.1 ± 2.6

Aspargine 6.0 ± 1.1 8.4 ± 3.4 3.7 ± 1.2 8.8 ± 1.8

Methionine 81.0 ± 16.5 65.9 ± 8.2 53.5 ± 6.2 75.2 ± 7.1

Cysteine 18.6 ± 6.1 27.0 ± 15.1 28.1 ± 9.7 26.7 ± 10.0

Phenylalanine 296 ± 60 283 ± 30 271 ± 36 279 ± 18

Tyrosine 14.7 ± 5.8 14.3 ± 4.0 12.9 ± 3.4 17.1 ± 3.2

Tryptophan 33.2 ± 15.4 33.2 ± 12.6 24.4 ± 8.4 16.2 ± 6.1

Histidine 1.99 ± 0.76 2.32 ± 0.96 2.95 ± 1.1 1.5 ± 0.3

Ornithine 11.7 ± 2.7 10.0 ± 2.0 10.7 ± 2.1 11.4 ± 2.0

WT-CNT WT-HFD KO-CNT KO-HFD

Lipid Metabolites15003 ±

Phosphate 14724 ± 801 1007 13232 ± 640 13590 ± 270

Diphosphate 1968 ± 402 5183 ± 2133 1636 ± 161 2930 ± 608

Phosphoglycerol 11517 ± 299 10969 ± 790 9647 ± 334 11729 ± 395

3-Phosphoglycerate 471 ± 84 438 ± 87 205 ± 27 204 ± 24

2-Phosphoglycerate 27.4 ± 4.3 23.4 ± 2.9 15.0 ± 1.5 13.0 ± 1.3

DHAP 7.47 ± 1.05 6.78 ± 0.46 5.53 ± 0.53 5.87 ± 0.60

Inositol 2964 ± 266 2030 ± 119 2283 ± 175 1940 ± 165

Myo-inositol Phosphate 345 ± 70 465 ± 109 258 ± 13 363 ± 74

Laurie Acid 37.5 ± 3.0 37.2 ± 3.6 32.6 ± 1.5 35.4 ± 2.0

Myristic Acid 87.8 ± 10.3 73.5 ± 5.5 87.1 ± 8.7 63.0 ± 5.5

Palmitaladic Acid 73.3 ± 17.0 40.4 ± 6.9 89.3 ± 23.5 24.4 ± 5.7

Palmitic Acid 6714 ± 1091 791 1 ± 1181 6285 ± 967 7838 ± 1014

Linoelic Acid 667 ± 143 759 ± 159 560 ± 145 572 ± 106

Oleic Acid 670 ± 138 851 ± 141 635 ± 171 662 ± 117

Elaidic Acid 120 ± 25 126 ± 28 175 ± 33 87 ± 18

Stearic Acid 2662 ± 462 4471 ± 682 2206 ± 344 3892 ± 559

Arachidonic Acid 56.0 ± 10.6 62.5 ± 15.4 46.4 ± 2.1 54.8 ± 6.8

1-Monooleoylglycerol 368 ± 85 498 ± 113 377 ± 129 475 ± 53

1-monostearylglycerol 1727 ± 367 2879 ± 502 1428 ± 337 2696 ± 289

1-Monopalmitoylglycerol 3808 ± 741 4606 ± 880 3645 ± 736 5011 ± 609

1-Monopalmitoylylglycerol 26.1 ± 6.0 23.6 ± 6.7 36.3 ± 10.8 16.5 ± 2.6

Cholesterol 1316 ± 113 1416 ± 163 1146 ± 126 1367 ± 109

WT-CNT WT-HFD KO-CNT KO-HFD

Nucleic Acid Metabolites

Xanthine 1133 ± 129 1183 ± 167 972 ± 83 940 ± 150

Adenosine 1.87 ± 0.33 1.82 ± 0.31 2.45 ± 0.26 2.16 ± 0.24

Inosine 394 ± 50 295 ± 50 276 ± 30 217 ± 52

Adenine 146 ± 13 153 ± 12 127 ± 4 167 ± 17

Uracil 42.7 ± 11.9 54.3 ± 9.0 21.6 ± 2.8 292.8 ± 98

[0083] Significant differences and interesting trends have been noted in several of these

metabolites as shown with respect to genotype (WT vs. KO) or the diet (regular diet vs. high-

fat diet) or both variables.

[0084] Based on the data disclosed herein, it appears that the KO mice are resistant to

high-fat diet-induced obesity. There are three possible explanations for this. The first

possibility is a decreased appetite leading to less consumption of food. A second possibility

is a lack of absorption of fat in the digestive system. Finally, the third possibility is increased

ability to burn more calories due to higher energy metabolism.

[0085] In the studied presented, the first two possibilities were eliminated, i.e, the KO

mice were not eating less food than the WT mice, and the KO mice had well-formed stools

with no evidence of steatorrhea. This leaves the third possibility, i.e., increased ability to

burn more calories or higher energy metabolism. To confirm this, the expression of key

genes in the WT and KO mice under basal or normal conditions, i.e., without feeding high-fat

diet, were examined.

[0086] Figure 14 shows the expression of two groups of key molecules involved in

energy metabolism in the brown fat, which is rich in mitochondria. These are the PPARs

(peroxisome proliferator-activated receptor), the nuclear receptors, involved in the regulation

of inflammation and energy homeostasis and thus represent important targets for obesity and

metabolic syndrome.

[0087] PPAR-a knockout mice are known to gain more adipose tissue mass as compared

to the wild type mice. PPAR-γ plays a role in adipocyte differentiation, and reduces

inflammatory gene expression. Less is known about the role of PPAR-δ in obesity, but it

stimulates fatty acid oxidation.

[0088] As shown in the left panel of Figure 14, PPAR-γ and PPAR-δ expression levels in

KO mice are significantly higher as compared to the WT mice, while the expression of

PPAR-a did not differ. Thus, the observed resistance of the KO mice to diet-induced obesity

is partly related to the differences in baseline expression of the PPAR isoforms in these mice

as compared to the WT mice.

[0089] Another potential group of molecules considered were the mitochondrial

uncoupling proteins (UCPs). UCPs are themogenic proteins and regulate energy expenditure.

They uncouple mitochondrial respiration for oxidative phosphorylation (formation of ATP),

resulting in dissipation of energy as heat, instead of converting into storage form of energy.

UCPs play a role in the development of obesity and type II diabetes mellitus. As shown in

the right panel of Figure 14, the basal expression levels of all three UCPs in the brown fat of

KO mice are significantly higher as compared to the expression levels in WT mice. This

advocates for the conclusion that KO mice are inherently capable of burning extra calories

consumed in the food.

[0090] The enlarged fat cells in the white adipose tissue in obesity recruit macrophages

(inflammatory cells) and promote inflammation, leading to elaboration of various cytokines

(inflammatory molecules) and chemical mediators. Accordingly, the inflammatory mediators

in the white fat of the WT and KO mice were examined after feeding high-fat diet for 16

weeks. As shown in the left panel of Figure 15, the expression levels of IL-6 (interleukin-6,

an inflammatory cytokine), MCP-1 (monocyte chemoattractant protein- 1 or CCL2) and its

receptor (CRR2), CD68 (monocyte/macrophage cluster differentiation 68 protein), as well as

F4/80 (a defining marker of murine macrophages) were significantly lower in the KO mice as

compared to the WT mice. This suggests a lesser degree of inflammation in the white

adipose tissue of KO mice versus WT mice after feeding high-fat diet.

[0091] During the study, the expression of insulin receptor substrate (IRS) 1 and 2 was

examined. These receptors transmit signals from the insulin receptor and insulin-like growth

factor-1 (IGF-1), and thus play an important role in metabolic pathways. Mice lacking IRS1

develop a mild diabetic phenotype, whereas the mice lacking IRS2 have a diabetic

phenotype. As shown in the right panel of Figure 15, the IRS1 expression was markedly

higher in the KO mice fed high-fat diet versus WT mice fed high-fat diet, whereas the

expression levels of IRS2 did not differ between these two genotypes. This finding suggests

that IRS1 contributes to the observed resistance of the KO mice to diet-induced obesity.

[0092] The above description of the disclosed embodiments is provided to enable any

person skilled in the art to make or use the invention. Various modifications to these

embodiments will be readily apparent to those skilled in the art, and the generic principles

described herein can be applied to other embodiments without departing from the spirit or

scope of the invention. Thus, it is to be understood that the description and drawings

presented herein represent a presently preferred embodiment of the invention and are

therefore representative of the subject matter which is broadly contemplated by the present

invention. It is further understood that the scope of the present invention fully encompasses

other embodiments that may become obvious to those skilled in the art and that the scope of

the present invention is accordingly not limited.

WHAT IS CLAIMED IS:

1. A method for treating or preventing diet-induced obesity in a subject by administering an

agent that blunts the expression or activity of the P2Y2 receptor in the subject.

2. The method of Claim 1, wherein the agent is a fusion protein.

3. The method of Claim 1, wherein the agent inhibits the P2Y2 receptor.

4. The method of Claim 3, wherein the agent is a selective P2Y2 receptor antagonist.

5. The method of Claim 4, wherein the selective P2Y2 receptor antagonist is a small non-

nucleic acid organic molecule.

6. The method of Claim 1, wherein the agent is an oligonucleotide.

7. The method of Claim 6, wherein the oligonucleotide is an antisense oligonucleotide.

8. The method of Claim 6, wherein the oligonucleotide is a ribozyme.

9. The method of Claim 7, wherein the oligonucleotide is an inhibitory RNA.

10. The method of Claim 9, wherein the inhibitor RNA is a small inhibitory RNA (siRNA).

11. A method for increasing energy metabolism in a cell by contacting the cell with an agent

that blunts the expression or activity of a P2Y2 receptor, thereby increasing the

metabolism in the cell.

12. The method of Claim 11 wherein in the agent is a fusion protein, polypeptide, a small

non-nucleic acid organic molecule, a small inorganic molecule or an oligonucleotide.

13. The method of Claim 12, wherein the oligonucleotide is an antibody.

14. The method of Claim 12, wherein the oligonucleotide is an antisense oligonucleotide.

15. The method of Claim 12, wherein the oligonucleotide is a ribozyme.

16. The method of Claim 12, wherein the oligonucleotide is an inhibitory RNA.

17. The method of Claim 16, wherein the inhibitory RNA is a small inhibitory RNA (siRNA).

18. The method of Claim 11, wherein the cell is an adipocyte cell.

19. A method for increasing energy metabolism in an adipocyte cell in a subject that is at

risk for or suffering from a disorder related to glucose metabolism, the method

comprising administering to the subject an agent that inhibits expression or activity of a

P2Y2 receptor, thereby increasing the metabolism in the cell.

20. The method of Claim 19, where in the agent is a polypeptide, a small non-nucleic acid

organic molecule, a small inorganic molecule or an antibody, an antisense

oligonucleotide, an inhibitory RNA or a ribozyme.

21. The method of Claim 20, wherein the inhibitory RNA is a small inhibitory RNA (siRNA).

22. A method for identifying a candidate agent that modulates expression or activity of the

P2Y2 receptor, comprising:

(a) providing a sample comprising the P2Y2 polypeptide or a nucleic acid encoding the

polypeptide;

(b) contacting the sample with a test compound under conditions in which the polypeptide

is active, the nucleic acid is expressed, or both;

(c) evaluating expression or activity of the P2Y2 polypeptide in the sample; and

(d) comparing the expression or activity of the P2Y2 polypeptide of (c) to the expression

or activity of the P2Y2 polypeptide in a control sample lacking the test compound,

wherein a change in the P2Y2 polypeptide expression or activity indicates that the test

compound is a candidate agent that can modulate the expression or activity of the

P2Y2 polypeptide.

23. The method of Claim 22, wherein the candidate agent selectively inhibits the P2Y2

receptor.

24. The method of Claim 22, wherein the candidate agent is a fusion protein.

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

Claims Nos. : 1-21

because they relate to subject matter not required to be searched by this Authority, namely:

Claims 1-21 pertain to a method for treatment of the human by therapy, and thus relate to a subject matter which thisInternational Searching Authority is not required, under Article 17(2)(a)(i) of the PCT and Rule 39. l(iv) of the Regulationsunder the PCT, to search.

□ Claims Nos. :because they relate to parts of the international application that do not comply with the prescribed requirements to such anextent that no meaningful international search can be carried out, specifically :

3 . Claims Nos. :because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

I IAs all required addtional search fees were timely paid by the applicant, this international search report covers all searchableclaims.

I IAs all searchable claims could be searched without effort justifying an additional fees, this Authority did not invite paymentof any additional fees.

I IAs only some of the required additional search fees were timely paid by the applicant, this international search report coversonly those claims for which fees were paid, specifically claims Nos.:

4 . I INo required additional search fees were timely paid by the applicant. Consequently, this international search report isrestricted to the invention first mentioned in the claims; it is covered by claims Nos. :

Remark on Protest | | The additional search fees were accompanied by the applicant's protest and, where applicable, thepayment of a protest fee.

I IThe additional search fees were accompanied by the applicant's protest but the applicable protestfee was not paid within the time limit specified in the invitation.

I INo protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet (2)) ( y 2009)

A . CLASSIFICATION OF SUBJECT MATTER

A61K 39/395(2006.01)i, A61K 48/00(2006.01)i, A61P 3/04(2006.01)i, A61P 3/00(2006.01)i

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)A61K 39/395; A61K 3 1/715; A61K 38/47; G01N 33/53; A61K 3 1/405; A61K 48/00; A61P 3/04; A61P 3/00

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searchedKorean utility models and applications for utility modelsJapanese utility models and applications for utility models

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)eKOMPASS(KXPO internal) & Keywords: purinoceptor, P2Y2, metabolism, obesity, diabetes

DOCUMENTS CONSIDERED TO BE RELEVANT

Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

W0 2005-100990 A2 (BAYER HEALTHCARE AG) 27 Oct ober 2005 22-24See abst ract ; page 33 , l ine 24 - page 34 , l ine V, page 62, l ines 20-35 ;page 64 , l ines 7-12 ; and c l aims 1-26 .

US 6372724 Bl (PELLEG, A. e t a l . ) 16 Apr i l 2002 22-24See abst ract and c l aims 1-23 .

US 2009-0297497 Al (KI SHORE , B. K. e t a l . ) 3 December 2009 22-24See abst ract and c l aims 1-34 .

I IFurther documents are listed in the continuation of Box C . See patent family annex.

* Special categories of cited documents: "T" later document published after the international filing date or priority"A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand

to be of particular relevance the principle or theory underlying the invention"E" earlier application or patent but published on or after the international "X" document of particular relevance; the claimed invention cannot be

filing date considered novel or cannot be considered to involve an inventive"L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone

cited to establish the publication date of another citation or other document of particular relevance; the claimed invention cannot bespecial reason (as specified) considered to involve an inventive step when the document is

"O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combinationmeans being obvious to a person skilled in the art

"P" document published prior to the international filing date but later document member of the same patent familythan the priority date claimed

Date of the actual completion of the international search Date of mailing of the international search report

28 January 2014 (28.01.2014) 28 January 2014 (28.01.2014)

Name and mailing address of the ISA/KR Authorized officer *»Korean Intellectual Property Office

-i Cheongsa-ro, Seo-gu, Daej eon Metropolitan City, CHOI, Sung Heefl 302-701, Republic of Korea

Facsimile No. +82-42-472-7140 Telephone No. +82-42-481-8740

Form PCT/ISA/210 (second sheet) (July 2009)

Information on patent family members PCT/US2013/066932

Patent document Publication Patent family Publicationcited in search report date member(s) date

WO 2005-100990 A2 27/10/2005 WO 2005-100990 A3 02/03/2006

US 6372724 Bl 16/04/2002 AT 470441 T 15/06/2010AU 1998-70985 B2 06/12/2001AU 7098598 A 20/10/1998AU 741713 B2 06/12/2001CA 2288821 Al 01/10/1998CA 2288821 C 14/10/2008DE 69841711 Dl 22/07/2010D 1011686 T3 11/10/2010EP 1011686 Al 28/06/2000EP 1011686 A4 19/03/2003EP 1011686 Bl 09/06/2010EP 1011686 B9 09/02/2011JP 2001-518118 A 09/10/2001US 2002-0055515 Al 09/05/2002us 6465441 B2 15/10/2002wo 98-42353 Al 01/10/1998

US 2009-0297497 Al 03/12/2009 AT 551054 T 15/04/2012CA 2585234 Al 08/06/2006EP 1807065 A2 18/07/2007EP 1807065 A4 06/05/2009EP 1807065 Bl 28/03/2012WO 2006-060079 A2 08/06/2006wo 2006-060079 A3 29/03/2007

Form PCT/ISA/210 (patent family annex) ( y 2009)